Detection of Neighboring Variants

ABSTRACT

The present invention relates to methods, kits, probes, and systems for distinguishing between nucleotide variants that are close in proximity on a gene. The methods, kits, probes, and systems can include the use of a small amplicon assay in combination with two unlabeled probes in a high resolution thermal melting analysis of a biological sample containing a locus of interest in order to discern between disease-causing and benign variants that are close in proximity on a gene within the biological sample. The present invention also relates to method of detecting a disease in a patient based on the patient&#39;s genotype by determining whether the patient has a disease-causing variant at a locus of interest. The signature melt curves produced by the unlabeled probe tests can be analyzed using HRMA software to distinguish between disease-causing and benign variants that are close in proximity on a gene within the biological sample.

This application is a divisional of U.S. Ser. No. 13/297,970, filed Nov.16, 2011, which is incorporated herein by reference in its entirety.

BACKGROUND

Field of the Invention

The present invention relates to methods, kits, primers, probes, andsystems for distinguishing between nucleotide variants that are close inproximity on a gene. More particularly, aspects of the present inventionrelate to methods, kits, primers, probes, and systems for using a smallamplicon assay in combination with unlabeled probes in conducting a highresolution thermal melting analysis of a biological sample containing alocus of interest in order to discern between disease-causing and benignvariants that are close in proximity on a gene. The present inventionalso relates to methods of detecting a disease in a patient based on thepatient's genotype by determining whether the patient has adisease-causing variant at a locus of interest on the patient's genome.

Description of the Background

Melt curve analysis or high resolution thermal melting is an importanttechnique for analyzing nucleic acids. In accordance with some methods,a double stranded nucleic acid is denatured in the presence of a dyethat indicates whether the two strands are bound or not. Examples ofsuch indicator dyes include non-specific binding dyes such as SYBR®Green I, whose fluorescence efficiency depends strongly on whether it isbound to double stranded DNA. As the temperature of the mixture isincreased, a reduction in fluorescence from the dye indicates that thenucleic acid molecule has melted, i.e., unzipped, partially orcompletely. Thus, by measuring the dye fluorescence as a function oftemperature, information is gained regarding the length of the duplex,the GC content or even the exact sequence. See, e.g., Ririe et al. (AnalBiochem 245:154-160, 1997), Wittwer et al. (Clin Chem 49:853-860, 2003),Liew et al. (Clin Chem 50:1156-1164 (2004), Herrmann et al. (Clin Chem52:494-503, 2006), Knapp et al. (U.S. Patent Application Publication No.2002/0197630), Wittwer et al. (U.S. Patent Application Publication No.2005/0233335), Wittwer et al. (U.S. Patent Application Publication No.2006/0019253), Sundberg et al. (U.S. Patent Application Publication No.2007/0026421) and Knight et al. (U.S. Patent Application Publication No.2007/0231799).

A number of commercial instruments exist that perform thermal melts onDNA. Examples of available instruments include the Idaho Technology HR-1high resolution melter and the Idaho Technology LightScanner highresolution melter. The HR-1 high resolution melter has a high resolutionfluorescent signal to noise ratio and temperature resolution. However,it suffers from a limitation that it can only analyze one sample at atime, and the sample container must be replaced manually. Replacement ofthe container for each test may contribute to run-to-run temperaturevariability. The LightScanner high resolution melter also has goodsignal and temperature resolution, and operates on a 96-well platesample container. A typical mode of operation for these analyzers is toapply heat to the sample(s) in a controlled manner to achieve a linearrise in temperature versus time. Simultaneously, a stable continuousfluorescence excitation light is applied, and emitted fluorescence iscollected continuously over fixed integration time intervals. Thefluorescence intensity data is converted from a time basis to atemperature basis based on the knowledge of the temperature ramp versustime.

In addition to such commercial instruments, microfluidic systems havealso been developed for performing thermal melt analysis. For example,Sundberg et al. (U.S. Patent Application Publication No. 2007/0026421)and Knight et al. (U.S. Patent Application Publication No.2007/0231799), each incorporated by reference herein, describe methods,systems, kits and devices for conducting binding assays using molecularmelt curves in microfluidic devices. Molecule(s) to be assayed can beflowed through microchannels in the devices where the molecule(s)optionally are exposed to additional molecules constituting, e.g.,fluorescence indicator molecules and/or binding partners of the moleculebeing assayed. The molecules involved are then heated (and/or cooled)and a detectable property of the molecules is measured over a range oftemperatures. From the resulting data, a thermal property curve(s) isconstructed which allows determination and quantification of the bindingaffinity of the molecules involved. Other microfluidic systems usefulfor thermal melting analysis are described in Hasson et al. (U.S. PatentApplication Publication No. 2009/0248349), Hasson et al. (U.S. PatentApplication Publication No. 2009/0318306), Hasson et al. (U.S. PatentApplication Publication No. 2009/0324037), Cao (U.S. Patent ApplicationPublication No. 2010/0233687) and Coursey (U.S. Patent ApplicationPublication No. 2011/0056926).

Although high resolution thermal melting is a useful tool forgenotyping, many genotyping assay attempts do not present a significantdifference in the melt curves of targeted variants, especially variantsthat are in close proximity. For example, prior techniques for CysticFibrosis (CF) testing using high resolution thermal melt analysis lackedthe ability to discern between benign and disease-causing variants inclose proximity in Exon 10 of the CFTR (cystic fibrosis transmembraneconductance regulator) gene.

Cystic fibrosis, also known as mucoviscidosis, is the most common lethalautosomal recessive disorder and the most common life-shorteninginherited diseases among the Caucasian population. Cystic Fibrosis iscaused by mutations of the CFTR gene. This disease affects multiplesystems and organs in the body, including the lungs, pancreas,intestines, and liver, and occurs in 1 in 2,500 Caucasian newborns(Rowntree and Harris, Annals of Human Genetics. 2003; 67:471-485).Currently, more than 30,000 children and young adults are affected byCystic Fibrosis in the United States. The ΔF508 mutation, a three basepair deletion that removes a phenylalanine residue at amino acidposition 508 (ΔF508), is the mutation occurring on the majority of CFchromosomes being found on 70%-75% of North American CF chromosomes(Kerem et al., Science. 1989; 245:1073-1080). This three base pairdeletion affects the cytoplasmic nucleotide-binding domain (NBD-1) andcauses severe dysfunction of chloride transportation a cross cellularmembranes.

Since ΔF508 was first identified in CF gene by Kerem and his colleaguesusing restriction fragment length polymorphisms (RFLP), more techniqueshave been used to explore CF mutations, such as a restriction map of thegenomic clone (Chou et al., J. Biol. Chem. 1991; 266:24471-24476),denaturing gel gradient electrophoresis (DGGE) using PCR (Pallares-Ruizet al. Human Reproduction. 1999; 14:3035-3040), allele-specific primersand fluorometry (Litia et al. Genome Res. 1992; 2:157-162), DNAsequencing (Kerem et al. Pediatrics. 1997; 100:1-6), and saturated dyeand melting analysis (Zhou et al. Clin. Chem. 2008; 54:1648-1656). Thesetechniques enhanced the understanding of the structures of the CFTR geneand CFTR mutations related to the CF disease, providing a promisingoutlook for early diagnosis and treatments to the CF patients.

However, it has been discovered that the benign variants F508C, I507V,and I506V that neighbor ΔF508/ΔI507, often present similar genotypepatterns and are mistakenly recognized as the disease-causing variantsΔF508 or ΔI507 (Desgeorges et al. Am J Hum Genet. 1994; 54:384-5). Thesubstitution of cysteine for phenylalanine 508 (F508C) and substitutionof valine for isoleucine 506 (I506V) have been reported to be homologousfor ΔF508 mutation and are difficult to be distinguished from ΔF508(Kobayashi et al. Am. J. Hum. Genet. 1990; 47:611-615). ΔI507, a threebase pair in frame deletion close to ΔF508 that results in the deletionof isoleucine, has been reported to present the same genotyping patternas I506V, a benign variant at the same location of ΔI507 (Johnson etal., J Mol Des 2007; 9:401-407).

Although commercial kits are available to detect CF mutations, thesekits do not make it possible to distinguish between all benign anddisease-causing mutations in Exon 10 in a rapid detection system(Johnson et. al., J. Mol. Diagn. 2007. 9:401-407). It is critical andnecessary for researchers, physicians, and diagnostic laboratories to beable to differentiate ΔF508 and ΔI507 from F508C, I507V, and I506V inorder to reliably detect and diagnose disease-causing genotypes.

Accordingly, there is a need in the art for reliable methods, kits,probes, and systems that will be useful in discerning between nucleotidesequence variants that present similar genotype patterns. Similarly,there is a need for methods, kits, probes, and systems for accuratelydetecting a disease in a patient when a disease-causing variant may bemistakenly recognized as a benign variant because the variants presentsimilar melting signatures.

SUMMARY

In one aspect, the present invention provides a method of distinguishingbetween at least two variants on a target nucleic acid having a locus ofinterest. In one embodiment, the method comprises (a) providing a firstaliquot of a nucleic acid having a locus of interest; (b) incubating thefirst aliquot of the nucleic acid with a limiting primer, an excessprimer, and a first probe that is designed to hybridize to the locus ofinterest on a target strand of the nucleic acid; (c) performingasymmetric PCR using the first aliquot to produce an excess of ampliconscorresponding to the target strand to which the first probe hybridizes,thereby producing a first probe element; (d) providing a second aliquotof the nucleic acid having a locus of interest; (e) incubating thesecond aliquot of the nucleic acid with the limiting primer, the excessprimer, and a second probe that is designed to hybridize to the locus ofinterest on the target strand, wherein the first probe differs insequence from the second probe; (f) performing asymmetric PCR using thesecond aliquot to produce an excess of amplicons corresponding to thetarget strand to which the second unlabeled probe hybridizes, therebyproducing a second probe element; (g) generating a first melting curvefor the first probe element in a first mixture with a saturating bindingdye by measuring fluorescence from the dye as the first mixture isheated; (h) generating a second melting curve for the second probeelement in a second mixture with the saturating binding dye by measuringfluorescence from the dye as the second mixture is heated; and (i)analyzing the first melting curve and the second melting curve todistinguish between the at least two nearby neighbor variants, wherein amelting signature curve of each of the at least two nearby neighborvariants is different in the first and second melting curves.

In one embodiment, one or both of the first and second probes can beunlabeled.

In one embodiment, the locus of interest is on a gene associated with adisease or disorder such as Cystic Fibrosis, Factor V Leiden, a RETproto-oncogene associated disease, lactase hemorrhagic telangiectasia,and hereditary hemorrhagic telangiectasia. Alternatively, the presentinvention can be used to identify or determine polymorphisms, such ashuman platelet antigens.

In another embodiment, the method steps can include (a) providing anamplicon having a locus of interest; (b) hybridizing a first unlabeledprobe to the locus of interest on a first portion of the amplicon toform a first probe element; (c) hybridizing a second unlabeled probe tothe locus of interest on a second portion of the amplicon to form asecond probe element, (d) generating a first melting curve for the firstprobe element; (e) generating a second melting curve for the secondprobe element; and (f) analyzing the first melting curve and the secondmelting curve to distinguish between the at least two nearby neighborvariants. In one embodiment, the melting curves are generated in thepresence of an intercalating or saturation dye by measuring thefluorescence as the probe element is heated.

In another aspect, the present invention provides a method ofdistinguishing between at least two variants on a target nucleic acidhaving a locus of interest comprising (a) mixing a first portion of atarget nucleic acid having a locus of interest with a first primer and asecond primer, the primers configured for amplifying the target nucleicacid having a locus of interest, and a first unlabeled probe; (b) inparallel, mixing a second portion of said target nucleic acid having alocus of interest with the first primer, the second primer, and a secondunlabeled probe; (c) simultaneously and asymmetrically amplifying thetarget nucleic acid having a locus of interest to generate ampliconsthat hybridize to the first unlabeled probe and the second unlabeledprobe to form a first probe element and a second probe element,respectively; (d) generating a first melting curve for the first probeelement; (e) sequentially or simultaneously generating a second meltingcurve for the second probe element; and (f) analyzing the first meltingcurve and the second melting curve to distinguish between the at leasttwo nearby neighbor variants.

In another aspect, the present invention provides a method of detectinga disease in a patient based on the patient's genotype and a prioriknowledge of benign and disease-causing variant gene sequencesassociated with the disease. In one embodiment, the method comprises thesteps of (a) obtaining a biological sample from the patient; (b)subjecting a first portion of the biological sample to asymmetric PCRinvolving a limiting primer, an excess primer, and a first probe toproduce a first probe-amplicon element; (c) subjecting a second portionof the biological sample to asymmetric PCR involving the limitingprimer, the excess primer, and a second probe to produce a secondprobe-amplicon element; (d) generating a first melting curve and asecond melting curve by subjecting the first and second probe-ampliconmelting elements to high resolution thermal melting analysis,respectively; (e) distinguishing between a benign variant and adisease-causing neighbor variant by analyzing the first melting curveand the second melting curve, wherein a probe melting signature curve ofthe benign variant and a probe melting signature curve of thedisease-causing variant in the first and second melting curves aredifferent; and (f) determining whether the patient has a disease-causingvariant.

In another aspect, the present invention provides a method of detectinga disease in a patient based on the patient's genotype and a prioriknowledge of benign and disease-causing variant gene sequencesassociated with the disease. In one embodiment, the method comprises thesteps of (a) obtaining a biological sample from a patient wherein thebiological sample includes a nucleic acid that has a locus of interest;(b) incubating a first portion of the sample with a limiting primer, anexcess primer, and a first unlabeled probe; (c) incubating a secondportion of the sample with the limiting primer, the excess primer, and asecond unlabeled probe; (d) subjecting each of the incubating first andsecond portions to asymmetric PCR in order to produce an excess of smallamplicons having the locus of interest; (e) once the limiting primer isexhausted, subjecting the first portion of the small amplicon to a firstunlabeled probe assay to produce a first melting curve and subjecting asecond portion of the small amplicon having a locus of interest to asecond unlabeled probe assay to produce a second melting curve; (f)distinguishing between a benign variant and a disease-causing neighborvariant by analyzing the first melting curve and the second meltingcurve, wherein a probe melting signature curve of the benign variant anda probe melting signature curve of the disease-causing variant in thefirst and second melting curves are different; and (g) determiningwhether the patient has a disease-causing variant.

In one embodiment, the disease to be detected can be a disease ordisorder such as Cystic Fibrosis, Factor V Leiden, a RET proto-oncogeneassociated disease, lactase hemorrhagic telangiectasia, or hereditaryhemorrhagic telangiectasia. Alternatively, the present invention can beused to identify or determine polymorphisms, such as human plateletantigens.

In another embodiment, the present invention provides a method ofdetecting a disease in a patient based on the patient's genotype and apriori knowledge of benign and disease-causing variant gene sequencesassociated with the disease. In one embodiment, the method comprises (a)obtaining a biological sample from a patient; (b) subjecting the sampleto asymmetric PCR to produce a small amplicon having a locus ofinterest; (c) subjecting a first portion of the small amplicon to afirst unlabeled probe assay to produce a first melting curve; (d)subjecting a second portion of the small amplicon having a locus ofinterest to a second unlabeled probe assay to produce a second meltingcurve; (e) distinguishing between a benign variant and a disease-causingneighbor variant by analyzing the first melting curve and the secondmelting curve, wherein a probe melting signature curve of the benignvariant and a probe melting signature curve of the disease-causingvariant in the first and second melting curves are different; and (f)determining whether the patient has a disease-causing variant.

In another embodiment, the method of detecting a disease in a patientbased on the patient's genotype and a priori knowledge of benign anddisease-causing variant gene sequences associated with the diseasecomprises (a) obtaining a biological sample from a patient; (b) dividingthe biological sample into a first portion and a second portion; (c)performing asymmetric PCR in order to produce a small amplicon having alocus of interest in each of the first portion and the second portion;(c) subjecting the first portion to a first unlabeled probe assay toproduce a first melting curve; (d) simultaneously or sequentiallysubjecting the second portion to a second unlabeled probe assay toproduce a second melting curve; (e) distinguishing between a benignvariant and a disease-causing neighbor variant by comparing the firstmelting curve and the second melting curve; and (f) determining whetherthe patient has a disease-causing variant.

In some embodiments, the present invention includes the use of a firstprobe and a second probe, wherein the first and second probes havesequences that differ from each other. In some embodiments, the firstand second probes are unlabeled.

In another aspect, the present invention provides a method ofdistinguishing between at least two nearby neighbor variants on anucleic acid having a locus of interest by (a) performing asymmetric PCRusing a primer pair and a first unlabeled probe; (b) performingasymmetric PCR using the primer pair and a second unlabeled probe,wherein the first unlabeled probe differs in sequence from the secondunlabeled probe; (c) generating a first melting curve for productsproduced in the asymmetric PCR using the first unlabeled probe in afirst mixture with a saturating binding dye by measuring fluorescencefrom the dye as the first mixture is heated; (d) generating a secondmelting curve for products produced in the asymmetric PCR using thesecond unlabeled probe in a second mixture with the saturating bindingdye by measuring fluorescence from said dye as the second mixture isheated; and (e) analyzing the first melting curve and the second meltingcurve to distinguish between the at least two nearby neighbor variants,wherein a melting signature curve of each of the at least two nearbyneighbor variants is different in the first and second melting curves.

In one embodiment, each of the asymmetric PCRs in steps (a) and (b)produce PCR products comprising small double-stranded amplicons andprobe/primer amplicons. In some embodiments, if two neighboringmutations cannot be clearly separated from melt signatures produced bythe probe/primer amplicons, the method further includes the step ofusing melt signatures produced by the small double-stranded amplicons todistinguish between the at least two nearby neighbor variants. Incertain embodiments, melting of the small double-stranded amplicons andthe probe/primer amplicons will provide thermal melt data for each typeof amplicon, which can be used to distinguish between the at least twonearby neighbor variants. Thus, the method may include the further stepof analyzing melt data from both the small double-stranded amplicons andfrom the probe/primer amplicons to distinguish between the at least twonearby neighbor variants.

In another aspect, the present invention provides a kit fordistinguishing between at least two variants on a target nucleic acidhaving a locus of interest and/or detecting a disease in a patient basedon the patient's genotype. In one embodiment, the kit comprises a firstunlabeled probe and a second unlabeled probe, wherein the first andsecond unlabeled probes have sequences that differ from each other. Inanother embodiment, the kit may further comprise primers for amplifyinga locus of interest on a target nucleic acid. In one embodiment, theprimers are selected for an asymmetric PCR amplification reaction. In afurther embodiment, the kit may also comprise instructions forperforming an amplification reaction and/or thermal analysis. The kitmay also comprise a dye that distinguishes between double stranded andsingle stranded nucleic acids. A suitable dye may be an intercalatingdye, a saturation dye or a double stranded DNA (dsDNA) binding dye.

In another aspect, the present invention provides a system fordistinguishing between at least two variants on a target DNA and/ordetecting a disease in a patient based on the patient's genotype. In oneembodiment, a system for distinguishing between at least two nearbyneighbor variants on a nucleic acid having a locus of interest accordingto the present invention can include (a) a microfluidic device having aplurality of sample loading zones, each of the sample loading zonesbeing configured to house a separate asymmetric PCR using a nucleic acidhaving a locus of interest; (b) a HRMA device, comprising a heatingelement, a fluorescence excitation light source and a fluorescencecollection aperture; and (c) a fluorescence derivative melting curveanalysis device configured to compare at least two melting curvesgenerated by the HRMA device so as to distinguish between at least twonearby neighbor variants on the nucleic acid having a locus of interest.

In one embodiment, the microfluidic device has at least two sampleloading zones that are loaded with a limiting primer, an excess primer,and an unlabeled probe. In certain embodiments, at least one of the atleast two sample loading zones that are loaded with an unlabeled probecontains a first unlabeled probe as the loaded unlabeled probe and atleast one of the at least two sample loading zones that are loaded withan unlabeled probe contains a second unlabeled probe as the loadedunlabeled probe. In some embodiments, the HRMA device is configured tothermally melt probe-amplicon elements obtained from asymmetric PCRs insaid sample loading zones, and to generate fluorescence derivativemelting curves for said probe-amplicon elements.

In some embodiments, the amplicon having a locus of interest is producedby mixing a target nucleic acid having a locus of interest with a firstprimer and a second primer, where the primers are designed to amplifythe target nucleic acid having a locus of interest, and amplifying thetarget nucleic acid having a locus of interest to generate an ampliconhaving the locus of interest. In some embodiments, the method steps areperformed simultaneously. In other embodiments, the method steps areperformed sequentially.

In some embodiments, unlabeled probes are designed to each have asequence that is complementary to the wild-type sequence. In someembodiments, a first primer and a second primer are each set close tothe targeted variants to reduce the amplicon size for high genotypingsensitivity. In some embodiments, unlabeled probes are designed so as tohave one or more base pair mismatches at the locus of interest when thetarget nucleic acid has a variant sequence. For example, in someembodiments, the unlabeled probes are designed to have one base pairmismatch at the locus of interest. In other embodiments, the unlabeledprobes may have a plurality of base pair mismatches, e.g., 3 bp mismatchin the case of a full codon deletion. In another embodiment, theunlabeled probes may have five or more base pair mismatches at the locusof interest. In some embodiments, an unlabeled probe has 2 to 5nucleotides at its 5′-end prior to the locus of interest, preferably 2or 3 nucleotides at its 5′-end prior to the targeted variants. In someembodiments, a second unlabeled probe has 5 nucleotides at its 5′-endprior to the targeted variants. In some embodiments, an unlabeled probeTm is less than about 5° C. lower than primer Tms and the difference ofa first primer's Tm and a second primer's Tm is less than about 1° C.

In some embodiments, a first unlabeled probe and a second unlabeledprobe are each 34 to 37 nucleotides in length. In some embodiments, afirst probe and a second probe are blocked at their 3′ ends.

In some embodiments, an amplicon having a locus of interest is producedusing asymmetric PCR. In some embodiments, a first primer is a limitingprimer in the asymmetric PCR. In some embodiments, a second primer is anexcess primer in the asymmetric PCR.

In some embodiments, the melting curves of the probe elements aregenerated in the presence of an indicator dye by measuring thefluorescence as the probe element is heated. As used herein, anindicator dye is a dye that distinguishes between double stranded andsingle stranded nucleic acids as described herein. In other embodiments,the melting curves of the probe assays are generated in the presence ofan indicator dye by measuring the fluorescence as the assay mixture isheated.

It is within the scope of the invention to obtain high resolutionthermal melting curves using at least two unlabeled probes for targetDNA sequences. It is also within the scope of the invention to designprobes and primers that are suitable for use in discerning betweenvariants in close proximity on the nucleic acid of interest. In oneembodiment, two or more unlabeled probes are designed to hybridize tothe same locus of interest on the target nucleic acid, but to producedifferent melting signature curves so as to provide meaningful data tobe used to distinguish between variants that present indistinguishablemelting curves using typical genotyping protocols.

Thus, in another aspect, the present invention also provides a method ofdesigning primers and probes that are useful for thermal melt analysisof a locus of interest that contains one or more benign variants and oneor more disease-causing variants that are in close proximity on thelocus of interest. In accordance with this aspect, the method comprises(a) selecting a locus of interest of a disease, in which the locus hasbenign variants and disease-causing variants in close proximity, (b)designing a pair of primers for use in asymmetric PCR, and (c) designingat least two probes for hybridizing to one strand of the locus ofinterest. In one embodiment, close proximity means within about 3-15nucleotides, preferably within 3-10 and more preferably within 3-7nucleotides of each other. In some embodiments the primers have a Tmdifference of less than about 1° C. and are selected to produce anamplicon of about 60 base pairs or longer upon amplification of thelocus of interest. In some embodiments, the PCR produces amplicons thatare between 70 and 130 bp in length. In certain embodiments, the PCRproduces amplicons that are between 80 and 100 bp in length. In someembodiments, the nucleotide sequence of each probe is complementary tothe wild-type sequence of the locus of interest. In other embodimentseach probe has a Tm that is less 5° C. lower than the Tms of theprimers. In some embodiments, each probe overlaps the nucleotidepositions of the one or more benign and one or more disease-causingvariants that are in close proximity on the locus of interest. In oneembodiment, the probes differ in length. In some embodiments, the probesdiffer in length as a result of the addition of residues at the 5′ endof the probe. In some embodiments, the probes each have a 3′ end blockto prevent extension.

The above and other aspects and features of the present invention, aswell as the structure and application of various embodiments of thepresent invention, are described below with reference to theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various embodiments of the presentinvention. In the drawings, like reference numbers indicate identical orfunctionally similar elements. Additionally, the left-most digit(s) ofthe reference number identifies the drawing in which the referencenumber first appears.

FIG. 1 is a gene map illustrating an embodiment of the invention showingthe mutations of ΔF508, ΔI507, F508C, I506V, I507V on a portion of CFTRExon 10.

FIG. 2 illustrates a schematic diagram of the primers, probe, and targetnucleic acid having a locus of interest.

FIG. 3 is a flow-chart showing a primer design process in accordancewith one embodiment of the present invention.

FIG. 4 illustrates a schematic diagram of the primers, probe, and aportion of CFTR Exon 10.

FIG. 5 is a high resolution melting curve profile for a portion of CFTRExon 10 obtained using a first unlabeled probe.

FIG. 6 is a high resolution melting curve profile for a portion of CFTRExon 10 obtained using a second unlabeled probe.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention has several embodiments and relies on patents,patent applications and other references for details known to those ofthe art. Therefore, when a patent, patent application, or otherreference is cited or repeated herein, it should be understood that itis incorporated by reference in its entirety for all purposes as well asfor the proposition that is recited.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A LaboratoryManual, PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, N.Y., Gait,Oligonucleotide Synthesis: A Practical Approach, 1984, IRL Press,London, Nelson and Cox (2000), Lehninger Principles of Biochemistry 3rdEd., W. H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5th Ed., W. H. Freeman Pub., New York, N.Y., all of whichare herein incorporated in their entirety by reference for all purposes.

As used herein, “homozygous” refers a genotype consisting of twoidentical alleles at a given locus.

As used herein, “heterozygous” refers to a genotype consisting of twodifferent alleles at a locus.

As used herein, “variant” refers to a permanent change in the DNAsequence of a gene, including mutations. Variants and mutations in agene's DNA sequence can alter the amino acid sequence of the proteinencoded by the gene.

As used herein, “benign variant” refers to an alteration in a genedistinct from the normal, wild-type allele that does not appear to havea deleterious physiological effect on the patient carrying the variant.

As used herein, “locus of interest” refers to a sequence of nucleotideson a nucleic acid/amplicon that is to be detected and/or analyzed. Thelocus of interest can be a site where variants/mutations are known tocause disease or predispose to a disease state. A locus of interest canbe a site of targeted nucleic acid variant within the context of a gene.

As used herein, “target nucleic acid” refers to one or more DNA or RNAmolecule(s) that is to be replicated, amplified, detected, and/oranalyzed. A “target nucleic acid” refers to deoxyribonucleic acid,ribonucleic acid or mixtures thereof. In addition, a “target nucleicacid” can further comprise non-natural nucleic acids. The target nucleicacid can be generated by any number of means. For example, it can begenerated from a cleavage reaction by a restriction enzyme or otherendo- or exonucleases. Alternatively, it can form as a result of aspecific or non-specific cleavage of a longer nucleic acid strand, andcan be generated enzymatically or chemically. The target nucleic acid ofthe present invention also contemplates fragments generated naturally invivo, by aged tissue, apoptotic cells, or the consequence of any othernatural, biological or chemical reaction that may generate nucleic acidfragments. The target nucleic acid may contain the locus of interest asa portion of its sequence or the locus of interest may make up theentire sequence of the target nucleic acid.

As used herein, “probe-amplicon elements” and “probe/primer amplicon”refer to the nucleic acid fragments or amplicons that are generatedduring PCR using a primer and a probe.

As provided throughout the specification, the steps of all of themethods described herein can occur in a sequential or simultaneousmanner, and alternatively some portion of the steps can occursimultaneously while others occur sequentially.

Some embodiments of the present invention utilize thermal melt curves todistinguish between at least two variants on a target nucleic acidhaving a locus of interest. Thermal melt curves of fluorescence havebeen used in the art to determine the melting temperature of a DNAstrand when denatured from the duplex state to the two separate singlestrands via a ramp increase in temperature. Typically, the meltingtemperature or Tm is defined to be the temperature at which 50% of thepaired DNA strands have denatured into single strands. Intercalatingdyes that fluoresce when bound to double stranded DNA and lose theirfluorescence when denatured are often used in measuring Tm. Typically,the negative derivative of fluorescence with respect to temperature(−dF/dT) has been used in the determination of Tm. In typical systems,the temperature at the peak −dF/dT is used as an estimate of the meltingtemperature Tm.

Melting curve analysis is typically carried out either in a stopped flowformat or in a continuous flow format. In one example of a stopped flowformat, melting curve analysis is done in a chamber to which the nucleicacid sample has been added. In an alternative stopped flow format, flowis stopped within a microchannel of a microfluidic device while thetemperature in that channel is ramped through a range of temperaturesrequired to generate the desired melt curve. In one example of acontinuous flow format, a melting curve analysis is performed byapplying a temperature gradient along the length (direction of flow) ofa microchannel of a microfluidic device. If the melting curve analysisrequires that the molecules being analyzed be subjected to a range oftemperatures extending from a first temperature to a second temperature,the temperature at one end of the microchannel is controlled to thefirst temperature, and the temperature at the other end of the length iscontrolled to the second temperature, thus creating a continuoustemperature gradient spanning the temperature range between the firstand second selected temperatures. An example of an instrument forperforming a melting curve analysis is disclosed in U.S. PatentApplication Publication No. 2007/0231799, incorporated herein byreference in its entirety.

The thermal melt data that is analyzed in accordance with aspects of thepresent invention is obtained by techniques well known in the art. See,e.g., Knight et al. (U.S. Patent Application Publication No.2007/0231799); Knapp et al. (U.S. Patent Application Publication No.2002/0197630); Wittwer et al. (U.S. Patent Application Publication No.2007/0020672); and Wittwer et al. (U.S. Pat. No. 6,174,670). Althoughthe present invention is applicable to the analysis of thermal melt dataobtained in any environment, it is particularly useful for thermal meltdata obtained in the microfluidic environment because of the need forgreater sensitivity in this environment.

Thermal melt data is typically generated by elevating the temperature ofa molecule or molecules, e.g., of one or more nucleic acids, for aselected period of time and measuring a detectable property emanatingfrom the molecule or molecules, wherein the detectable propertyindicates an extent of denaturation of the nucleic acid. This period oftime can range, for example, from about 0.01 second through to about 1.0minute or more, from about 0.01 second to about 10 seconds or more, orfrom about 0.1 second to about 1.0 second or more, including all timeperiods in between. In one embodiment, heating comprises elevating thetemperature of the molecule or molecules by continuously increasing thetemperature of the molecule or molecules. For example, the temperatureof the molecule(s) can be continuously increased at a rate in the rangeof about 0.1° C./second to about 1° C./second. Alternatively, thetemperature of the molecule(s) can be continuously increase at a slowerrate, such as a rate in the range of about 0.01° C./second to about 0.1°C./second, or at a faster rate, such as a rate in the range of about 1°C./second to about 10° C./second. The heating can occur throughapplication of an internal or an external heat source, as is known inthe art.

The actual detection of a change(s) in a physical property of themolecules can be detected in numerous methods depending on the specificmolecules and reactions involved. For example, the denaturation of themolecules can be tracked by following fluorescence or emitted light frommolecules in the assay. The degree of, or change in, fluorescence iscorrelational or proportional to the degree of change in conformation ofthe molecules being assayed. Thus, in some methods, the detection of aproperty of the molecule(s) comprises detecting a level of fluorescenceor emitted light from the molecules(s) that varies as a function ofrelative amounts of binding. In one configuration, the detecting offluorescence involves a first molecule and a second molecule, whereinthe first molecule is a fluorescence indicator dye or a fluorescenceindicator molecule and the second molecule is the target molecule to beassayed. In one embodiment, the fluorescence indicator dye orfluorescence indicator molecule binds or associates with the secondmolecule by binding to hydrophobic or hydrophilic residues on the secondmolecule. The methods of detecting optionally further comprise excitingthe fluorescence indicator dye or fluorescence indicator molecule tocreate an excited fluorescence indicator dye or excited fluorescenceindicator molecule and discerning and measuring an emission or quenchingevent of the excited fluorescence indicator dye or fluorescenceindicator molecule. See, e.g., Boles et al. (U.S. Patent ApplicationPublication No. 2009/0112484); Cao et al. (U.S. Patent ApplicationPublication No. 2009/0112481); Knight et al. (U.S. Patent ApplicationPublication No. 2007/0231799), which are incorporated herein byreference.

Several techniques exist for the measurement of the denaturation of themolecules of interest, and any of these can be used in generating thedata to be analyzed in accordance with aspects of the present invention.Such techniques include fluorescence, fluorescence polarization,fluorescence resonance energy transfer, circular dichroism and UVabsorbance. Briefly, the fluorescence techniques involves the use ofspectroscopy to measure changes in fluorescence or light to track thedenaturation/unfolding of the target molecule as the target molecule issubjected to changes in temperature. Spectrometry, e.g. viafluorescence, is a useful method of detecting thermally induceddenaturation/unfolding of molecules. Many different methods involvingfluorescence are available for detecting denaturation of molecules (e.g.intrinsic fluorescence, numerous fluorescence indicator dyes ormolecules, fluorescence polarization, fluorescence resonance energytransfer, etc.) and are optional embodiments of the present invention.These methods can take advantage of either internal fluorescentproperties of target molecules or external fluorescence, i.e. thefluorescence of additional indicator molecules involved in the analysis.See, e.g., Cao (U.S. Patent Application Publication No. 2010/0233687),incorporated herein by reference in its entirety.

One method of measuring the degree of denaturation/unfolding of thetarget molecule is through monitoring of the fluorescence of dyes ormolecules added to the microfluidic device along with the targetmolecule and any test molecules of interest. A fluorescence dye ormolecule refers to any fluorescent molecule or compound (e.g., afluorophore) which can bind to a target molecule either once the targetmolecule is unfolded or denatured or before the target moleculeundergoes conformational change by, for example, denaturing and whichemits fluorescent energy or light after it is excited by, for example,light of a specified wavelength.

One dye type typically used in the microfluidic devices is one thatintercalates within strands of nucleic acids. An example of such a dyeis ethidium bromide. An exemplary use of ethidium bromide for bindingassays includes, for example, monitoring for a decrease in fluorescenceemission from ethidium bromide due to binding of test molecules tonucleic acid target molecules (ethidium bromide displacement assay).See, e.g., Lee et al. (J Med Chem 36:863-870, 1993). The use of nucleicacid intercalating agents in measurement of denaturation is known tothose in the art. See, e.g., Haugland (Handbook of Fluorescent Probesand Research Chemicals, 9^(th) Ed., Molecular Probes, Inc., Eugene,Oreg., 2002).

Dyes that bind to nucleic acids by mechanisms other than intercalationare also typically employed in thermal melt analysis. For example, dyesthat bind the minor groove of double stranded DNA can be used to monitorthe molecular unfolding/denaturation of the target molecule due totemperature. Examples of suitable minor groove binding dyes are theSYBR® Green family of dyes sold by Molecular Probes Inc. (Eugene, Oreg.,USA). See, e.g., Haugland (Handbook of Fluorescent Probes and ResearchChemicals, 9^(th) Ed., Molecular Probes, Inc., Eugene, Oreg., 2002).SYBR® Green dyes will bind to any double stranded DNA molecule. When aSYBR Green dye binds to double stranded DNA, the intensity of thefluorescent emissions increases. As more double stranded DNA aredenatured due to increasing temperature, the SYBR® Green dye signal willdecrease. Other suitable dyes are LCGreen® Plus sold by IdahoTechnology, Inc. (Salt Lake City, Utah, USA), SYTO® 9 sold by InvitrogenCorp. (Carlsbad, Calif.) and, Eva Green® sold by Biotium Inc. (Hayward,Calif.). Further examples of dyes include SYBR® Green I (BIORAD,Hercules, Calif.), ethidium bromide, SYBR® Gold (INVITROGEN), PicoGreen, TOTO-1 and YOYO-1. It is within the skill of persons of ordinaryskill in the art to select a suitable dye.

In accordance with aspects of the present invention, methods, kits,primers, probes, and systems for distinguishing between nucleotidevariants that are close in proximity on a gene are provided. In oneexemplary embodiment, the method of the present invention is useful fordistinguishing between nucleotide variants that are in close proximityof the CFTR gene, such as those found in Exon 10, in order to reliablydetect and diagnose disease-causing CF genotypes. The present inventioncan also be suitably applied to distinguish between other nucleotidevariants that are close in proximity, e.g., mutations of G551D and R553Xof Exon 11 on the CFTR gene. G551D, a disease-causing mutation of asingle base change from G to A (position of c.1652), has 4% frequencyamong CF patients, and is only 4 bp from R553X, another disease-causingmutation of a single base change from C to T. Both mutations have beengenotyped with the dual primer (one of which is limited and one of whichis in excess) and unlabeled probe design described herein. Similarly,the present invention can also be used to identify G551S, a mutationthat can remarkably reduce single-channel open probability, and isanother single base mutation from G to A on the position of c.1651. Itis theorized that distinguishing G551D and R553X from G551S may requirethe use of an additional unlabelled probe. Thus, it is within the scopeof the present invention that multiple assays, each using a limitingprimer and an excess primer in combination with an unlabeled probe canbe used to distinguish between two or more nucleotide variants. Forinstance, in some cases, distinguishing between three nucleotidevariants may require three assays, each having its own limiting primerand an excess primer in combination with an unlabeled probe.

FIG. 1 illustrates a gene map of one embodiment of the invention showingthe mutations of ΔF508, ΔI507, F508C, I506V, I507V on a portion of CFTRExon 10. ΔI507, a three base pair in frame deletion close to ΔF508,results in the deletion of isoleucine. ΔF508 can occur due to deletionof either TCT or CTT. Both 3 base pair deletions of TCT or CTT lead tothe same DNA sequence -ATTTG when ΔF508 mutation presents. ΔI507, on theother hand, presents a much less severe phenotype compared to ΔF508.There are two consecutive isoleucine codons (ATC) between nucleotides1515 and 1522 having the sequence: A(1516)T(1517)C(1518)A(1519)T(1520)C(1521). Deletion of any 3 base pairs in the rangeof between 1516 and 1521 would produce the same sequence. The benignvariants, F508C, I507V and I506V, are located within the same region onExon 10 as ΔI507 and ΔF508. Identification of disease-causing variantsΔF508/ΔI507 is hindered by the presence of neighboring benign variants.The presence of variants in close proximity results in incorrectclinical diagnoses because the melting signatures for benign anddisease-causing variants that are in close proximity are often notreliably discernible.

FIG. 2 is a schematic diagram illustrating an embodiment of the presentinvention. A limiting primer (200) for amplification in the 5′-3′direction of a target double stranded nucleic acid (210) havingmutations of interest (220-224) at a locus of interest (230) and anexcess primer (240) for amplification of the target oligonucleotide(210) in the 3′-5′ direction are used to produce small amplicons. Anunlabeled probe (250) having a blocker (260) at its 3′ end, and designedto hybridize to the reverse strand of the target nucleic acid (210) atthe locus of interest (230), is used for high resolution thermal meltinganalysis. One or more additional unlabeled probes (not shown), which arealso designed to hybridize to the reverse strand of the target nucleicacid (210) at the locus of interest (230), but having a sequence that isdifferent from the first unlabeled probe (250), are used to obtain oneor more additional high resolution thermal melting curves. The schematicmutations of interest (220-224) include benign variants (220, 222 and224), as well as disease causing variants (221 and 223), all of whichare located in close proximity to each other.

In accordance with a method of the present invention, an amplicon isprovided which contains the locus of interest. The amplicon can beproduced by any method that results in the amplification of a targetnucleic acid. Amplification reactions are well known in the art, and theskilled artisan can readily use any suitable amplification reaction. PCRis perhaps the most well-known of a number of the differentamplification techniques. In one embodiment, asymmetric PCR is utilizedto generate the amplicon. As is well known in the art, asymmetric PCRuses one primer in a limiting concentration and the other primer inexcess concentration to preferentially amplify one DNA strand in adouble-stranded DNA template. It is typically used in sequencing andhybridization probing where amplification of only one of the twocomplementary strands is required. PCR is carried out as usual, but withan excess of the primer for the strand targeted for amplification.Because of the slow (arithmetic) amplification later in the reactionafter the limiting primer has been used up, extra cycles of PCR arerequired. See Innis et al. (Proc Natl Acad Sci USA 85:9436-9440, 1988).A recent modification on this process, known asLinear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer witha higher Tm rather than the excess primer to maintain reactionefficiency as the limiting primer concentration decreases mid-reaction.See Pierce and Wangh (Methods Mol Med 132:65-85, 2007). In a preferredembodiment, basic asymmetric PCR is utilized to generate an ampliconhaving the locus of interest.

Thus, in one embodiment of a method in accordance with the presentinvention, asymmetric PCR is used to preferentially amplify one DNAstrand of a target double stranded DNA template in order to produceamplicons having the locus of interest in order to distinguish betweenat least two variants on a target DNA. In a non-limiting embodiment,small amplicons having 45-130 base pairs, preferably 60-100 base pairs,are produced. Small amplicons usually have the advantages of efficientPCR, short cycles and distinguishable Tm separations for homozygote anda unique melting shape for heterozygotes. However, in many cases, thethermal melting of such small amplicons will not itself be able todifferentiate nearby neighbor mutations/variants, e.g., CFTR ΔF508 andΔI507 from F508C, I506V and I507V, due to the complexity of such closeproximity variants/mutations. In accordance with the present invention,a plurality of unlabeled probes is utilized to distinguish the variantsin the amplicon. As used herein, a plurality of probes refers to atleast two probes. In one embodiment, the unlabeled probes have differentlengths and/or different target specificities. Design considerations forthe primers to produce the small amplicon and for the plurality ofprobes are described in further detail herein.

According to one aspect of the present invention, the method includeshybridizing different portions of a small amplicon produced byasymmetric PCR with different unlabeled probes. A single portion of thesmall amplicon hybridizes with a single unlabeled probe to produce aprobe-amplicon element, such that a probe-amplicon element is producedfor each of the plurality of unlabeled probes. For example, if twounlabeled probes are utilized, a first portion of the ampliconhybridizes with a first unlabeled probed to produce a firstprobe-amplicon element and a second portion of the amplicon hybridizeswith a second unlabeled probed to produce a second probe-ampliconelement. The probe-amplicon element can be formed in the presence of anindicator dye, such as any of those described herein and well known tothe skilled artisan. The use of the unlabeled probes described hereinproduces unique melting signatures at lower temperature regions from thesmall amplicon Tm. Melting signature curves can be obtained bysubjecting the dyed unlabeled probe-amplicon elements to high resolutionthermal melting analysis as described herein. The method of the presentinvention, e.g., the use of a small amplicon and specially designedprimers and probes, is capable of distinguishing variants that are inclose proximity on a target nucleic acid.

In one embodiment, a biological sample containing a target nucleic acidhaving a locus of interest is subjected to asymmetric PCR to generatesmall amplicons having the locus of interest. The asymmetric PCR can beperformed in any suitable instrument for performing PCR, includingthermal cyclers and microfluidic devices well known to the skilledartisan. The reaction mixture containing the small amplicons having alocus of interest is divided into a plurality of portions to be keptseparated. Each of the separate small amplicon portions is involved inhybridization with one of the plurality of unlabeled probes so that aplurality of amplicon-probe hybridization reactions occurs sequentiallyor simultaneously to produce a plurality of probe-amplicon elements. Inone embodiment, each of the plurality of unlabeled probes is perfectlycomplementary to the wild-type sequence of the reverse strand of thetarget nucleic acid, but the individual unlabeled probes each have adifferent length.

In another embodiment of the invention, a biological sample containing atarget nucleic acid having a locus of interest is divided into two ormore portions. Each portion of the biological sample is thensimultaneously or sequentially subjected to asymmetric PCR to generatesmall amplicons having the locus of interest, which are then subjectedto hybridization with unlabeled probes so that a plurality ofamplicon-probe hybridization reactions can be performed sequentially orsimultaneously. In one embodiment, primers and an unlabeled probe areadded to a divided portion of the biological sample for amplification,annealing and extension. Each of the individual divided sample portionsis used for amplification in the presence of one of the unlabeled probesso that a plurality of amplification reactions is performed sequentiallyor simultaneously to produce a plurality of probe-amplicon elements. Inone embodiment, each of the plurality of unlabeled probes is perfectlycomplementary to the wild-type sequence of the reverse strand of thetarget nucleic acid, but the individual unlabeled probes each have adifferent length.

In another aspect, a system for distinguishing between at least twonearby neighbor variants on a nucleic acid having a locus of interestaccording to the present invention is provided which may comprise (a) amicrofluidic device comprising a plurality of sample loading zones, eachof the sample loading zones being configured to house a separateasymmetric PCR using a nucleic acid having a locus of interest, whereinthe microfluidic device comprises at least two sample loading zones thatare loaded with a limiting primer, an excess primer, and an unlabeledprobe, and wherein at least one of the at least two sample loading zonesthat are loaded with an unlabeled probe contains a first unlabeled probeas the loaded unlabeled probe and at least one of the at least twosample loading zones that are loaded with an unlabeled probe contains asecond unlabeled probe as the loaded unlabeled probe; (b) a HRMA device,comprising a heating element, a fluorescence excitation light source anda fluorescence collection device, configured to thermally meltprobe-amplicon elements obtained from asymmetric PCRs in the sampleloading zones, and to generate fluorescence derivative melting curvesfor the probe-amplicon elements; and (c) a fluorescence derivativemelting curve analysis device configured to compare at least two meltingcurves generated by the HRMA device so as to distinguish between atleast two nearby neighbor variants on the nucleic acid having a locus ofinterest. Examples of microfluidic devices that can be used according tothe system of the present invention are described in Hasson et al. (U.S.Patent Application Publication No. 2010/0191482), as well as othersdisclosed herein, which are incorporated herein by reference in theirentirety. Examples of possible HRMA devices configured for fluorescencemeasurement are illustrated in U.S. Patent Application Publication Nos.2008/0003593 and 2009/0324037 and U.S. Pat. No. 7,629,124, as well asothers disclosed herein, which are incorporated herein by reference intheir entirety. In one exemplary embodiment, a fluorescence derivativemelting curve analysis device can be an appropriately programmedcomputer that can render and compare at least two melting curvesgenerated by the HRMA device as disclosed herein. Conventional highresolution thermal melt software well known to the skilled artisan,including Genotype Determinator and Melt Viewer, can be used torecognize and compare the probe melting signatures of each genotype fortargeted amplicons.

In one embodiment, amplification of the biological sample having a locusof interest and annealing and extension of the small amplicons producedby amplification are performed in a microfluidic device. In oneembodiment, the microfluidic device has a plurality of channels foramplification, annealing and extension of one or more biologicalsamples, or one or more portions of one biological sample, in parallel.In one embodiment, the microfluidic device has a plurality of wells forloading the biological sample onto the device. In another embodiment,the microfluidic device has a first part for performing PCRamplification and a second part for performing thermal melt analysis. Adescription of PCR amplification, and examples of microfluidic devicesincluding thermal control elements for PCR amplification and thermalmelt analysis are provided in U.S. Patent Application Publication Nos.2009/0248349, 2009/0318306, 2009/0324037, 2010/0233687 and 2011/0056926,the entire disclosures of which are incorporated herein by reference.

Embodiments of the present invention can be used in a variety ofinstruments but are particularly useful in PCR and thermal melt systemsthat perform in vitro diagnostics. Embodiments of the present inventionmay be used in devices that are intended for thermal melt of samples(diagnostics) as well as other heaters and sensors within the instrumentthat perform entirely different functions (e.g., sample prep or PCR).Examples of microfluidic devices known in the art include, but are notlimited to, Chow et al. (U.S. Pat. No. 6,447,661), Kopf-Sill (U.S. Pat.No. 6,524,830), Spaid (U.S. Pat. No. 7,101,467), Dubrow et al. (U.S.Pat. No. 7,303,727), Schembri (U.S. Pat. No. 7,390,457), Schembri (U.S.Pat. No. 7,402,279), Takahashi et al. (U.S. Pat. No. 7,604,938), Knappet al. (U.S. Patent Application Publication No. 2005/0042639), andHasson et al. (U.S. Patent Application Publication No. 2010/0191482), aswell as others disclosed herein. Each of these patents or patentapplication publications is incorporated herein by reference.

In one or more embodiments of the invention, after amplification,annealing and extension, the probe-amplicon elements are subjected tosaturation dyes and high resolution melting analysis in order togenerate melting curves. Each probe-amplicon element can produce aunique signature melting curve because the length and/or target of theunlabeled probes used for hybridization in each run is different,thereby resulting in a different melt signature. When the targetedsequence contains disease-causing mutations/variants or nearbyneighboring benign variant(s), during melting, the melting signatures ofthese mutations/variants can be definitively displayed on the probemelting region on the melting curve. Accordingly, analysis of thegenerated melting curves using two or more unlabeled probes for thetarget nucleic acid having a locus of interest makes it possible todiscern between variants that are in close proximity on the targetsequence. An example of one possible fluorescence measurement system isillustrated in U.S. Patent Application Publication Nos. 2008/0003593 and2009/0324037 and U.S. Pat. No. 7,629,124, which are incorporated hereinby reference in their entirety.

In order to distinguish among the variants that are close in proximityon the target nucleic acid, specially designed primers and unlabeledprobes are used to produce the small amplicons and for the thermal meltanalysis. FIG. 3 illustrates a flow chart for a method of designingprimers according to one embodiment of the present invention. In oneembodiment for designing primers, mutation information can be obtained(310), e.g., from the American College of Medical Genetics (ACMG)(www.acmg.net/Pages/ACMG_Activities/stds-2002/cf.htm). From thisinformation, DNA sequences of interest for primer design can be selected(320). In an exemplary embodiment, the DNA sequences can be selectedfrom the CFTR gene database (www.genet.sickkids.on.ca/PicturePage.html).In one embodiment, the DNA sequences are selected so that themutation(s) of interest is located in about the middle of the sequenceof interest, i.e., the sequence containing the mutation(s) of interest.The length of the selected DNA sequence can vary, but may be for exampleseveral hundred bps. In one embodiment, the DNA sequence may be about150-500 bp in length. In an embodiment, the DNA sequence may be about200-400 bp in length. In an embodiment, the DNA sequence may be 300 bpin length. Once selected, each DNA sequence of interest is compared withother sequences to define regions of local similarity between sequencesfor each of the DNA sequences of interest (330). In one embodiment, theother sequences can be found on NCBI sequence databases and blast searchtools such as BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi) and Human BLAST(genome.ucsc.edu/cgi-bin/hgBlat?command=start&org=Human&db=hg18&hgsid=151501082)can be used for comparisons. Primer design can then be implemented,e.g., by using primer design software. Examples of such softwareinclude, but are not limited to, Primer 3 software(frodo.wi.mit.edu/primer3/) or SNP Wizard software (courtesy of CarlWittwer, University of Utah) (340) to produce primer sequences (350).Sequence similarity of primers output by the software and othersequences within the same genome are compared using tools such as BLASTand Human BLAST (360) to obtain sequence alignments (370). If the primersequences are the same as other sequences within the same genome onmultiple locations, e.g. on different chromosomes, or on the samechromosome, this primer is rejected (380). If the primer sequence is aunique sequence, which, in a preferred embodiment, is 100% differentfrom any other sequences, this primer and/or probe are accepted (390).Once primer pairs are designed as described above and accepted (391),the primer pairs are checked with, for example, In-Silico PCR tool forPCR prediction for PCR conditions and products. An example of anIn-Silico PCR tool includes, but is not limited to,(genome.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=hg18&org=Human&wp_f=&wp_r=&wp_size=4000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=151501082) (392). Theaccepted sequences can be examined for theoretical melting prediction(393). Examples of melt prediction software include, but are not limitedto, UMelt software (courtesy of Carl Wittwer, University of Utah,www.dna.utah.edu/umelt/um.php) and Poland Melt Prediction software(www.biophys.uni-duesseldorf.de/local/POLAND/). In some embodiments, thewild type sequence and the mismatched sequence of the targeted mutationscan be checked with both programs. The predicted results can becompared, recorded, and later used for comparing with experimental dataas references. Once the primer sequences have passed the above criteriaand checks, they can be tested for the assay feasibility, includingsensitivity, specificity, and reproducibility of the assay (394).

In some embodiments, a primer pair is designed so that a forward primeris set as the limiting primer and a reverse primer is set as the excessprimer in the asymmetric PCR. In other embodiments, a primer pair isdesigned so that a forward primer is set as the excess primer and areverse primer is set as the limiting primer in the asymmetric PCR. Eachprimer is set close to the targeted variant region so that the ampliconsize can be reduced for high genotyping sensitivity. In someembodiments, the primers are set from 20-80 nucleotides, preferably25-70 nucleotides, more preferably 30-60 nucleotides from the targetedvariant region. In some embodiments, the difference of the firstprimer's Tm and the second primer's Tm is less than about 1° C. In someembodiments, the Tm of each prime is, independently, between 52° C. and65° C. In some embodiments, the primers are independently from about 15to about 30 bp in length. In some embodiments, the primers areindependently from 18 to 25 bp in length. In certain embodiments theprimers are independently from 18-22 bp in length. In some embodiments,each primer has GC content between about 40-60%, independently. Incertain embodiments, the primers are designed to avoid having more thanthree G or C nucleotides in the last 5 bases at the 3′ end of theprimers. In some embodiments, the primers are designed to have a maximumof four di-nucleotide repeats.

In certain embodiments, one or more unlabeled probes are designed thathave a sequence that is complementary to the wild-type sequence. In oneembodiment, the probes are designed under the same criteria as theprimers for identical PCR conditions. After identifying the mutation andthe locus of interest on a gene, probe design can be performed byplacing the probe within the locus of interest region. The sequence canbe aligned using the BLAST or HumanBlast tools previously mentioned.Using these tools, any probe sequences that are homologous to otherregions of unrelated sequences should be excluded. In some embodiments,the probe is designed so as to have its Tm be less than about 70° C. Incertain embodiments, the probe is designed so as to have its Tm be lessthan the Tm of the primers. In preferred embodiments, probes aredesigned so that the targeted nucleic acid variants are located atapproximately the middle of the length of the probe.

In some embodiments, the unlabeled probes are perfectly complimentary tothe wild-type sequence on the reverse strand of the target nucleic acid.In other embodiments, the unlabeled probes are perfectly complimentaryto the wild-type sequence on the forward strand of the target nucleicacid. Thus, in individuals having mutations/variants at the locus ofinterest, the unlabeled probes will have one or more single base pairmismatch(es) at the corresponding locus. This design is to increase theassay sensitivity and specificity of all of the mutations/variants atthe locus of interest on the target nucleic acid. Thus, in oneembodiment, the length of the unlabeled probe is designed so as to havesufficient sensitivity and specificity to the mutations/variants withinthe locus of interest. In some embodiments, an unlabeled probe can bedesigned to have 2 to 5 nucleotides at its 5′-end prior to the locus ofinterest, preferably 2 or 3 nucleotides at its 5′-end prior to thetargeted variants. In some embodiments, a second unlabeled probe has 5to 8 nucleotides at its 5′-end prior to the targeted variants. In someembodiments, a first probe and a second probe are used for generatingmelting curves for the same biological sample, wherein the probes havedifferent lengths. The length of the unlabeled probes can be varieddepending on the melting characteristics of the targeted amplicons.

Generally, the longer the probe length, the stronger the probe meltsignals. In an exemplary embodiment, when the probe length is about ⅓ ofthe amplicon size, the probe melt signals are significantly increasedand easy to identify each targeted mutation. When the probe size isgreater than ⅓ of amplicon size, the probe melt signal is remarkablystrong and the fine separations between nearby mutations and signaturemelt shapes can be easily obtained. However, in some embodiments, theprobe length can be less than ⅓ of the amplicon size.

In one embodiment, the probes are designed to have between 25-50nucleotides, preferably between 30-40 nucleotides, more preferablybetween 33-39 nucleotides. The probe length is selected so to increasethe probe melting signal strength. In certain exemplary embodiments, oneunlabeled probe can be about 34 nucleotides in length while anotherunlabeled probe can be about 37 nucleotides in length. In someembodiments, a first probe and a second probe are each blocked at the 3′end to prevent extension of the probe. The 3′ end may be blocked withany suitable blocker, for example, a 3′ C6-amino blocker, a 3′phosphorylation blocker, or any other suitable 3′ blockers. During probedesign, the range of optimal Tms of probe is about 2-6° C. lower thanprimer Tms. In one embodiment, the probe Tm is less than about 5° C.lower than primer Tms. In another embodiment, the probe Tm is about 3°C. lower than primer Tms. Unlabeled probe design has been used todetermine complex mutations on other genes. In some embodiments, thepresent invention can be used for Factor V Leiden genotyping, humanplatelet antigens, the RET proto-oncogene, lactase and hereditaryhemorrhagic telangiectasia genotypings. In certain embodiments, careshould be taken to avoid designing primers and probes that are the sameat multiple locations.

One of the unique features of the present invention is the availabilityof multiple sources of data to distinguish between neighboringmutations. If two neighboring mutations cannot be clearly separated fromthe probe melt curves, then the amplicon melt curves should be used toidentify these mutations. One of skill in the art will recognize thatthe assay procedure described herein results in the production of smalldouble-stranded amplicons (from the primer/primer PCR) and singlestranded probe/primer amplicons (from the unlabeled probe and excessprimer PCR). Melting of the amplicons from the assay will providethermal melt data for each type of amplicon, and therefore the data fromboth the small double-stranded amplicons and from the probe/primeramplicons can be used to distinguish between neighboring mutations. Forinstance, the low temperature region is the probe/primer melt region,which reflects fluorescence signal levels changes during the temperaturetransition when the probe was melted off from the amplicon. The highertemperature melt region presents the fluorescence signal changes duringthe double stranded amplicon melt. Usually, if the amplicon size issmall, the changes in the double stranded amplicon melt curves can helpto separate the variants that may be difficult to distinguish in theprobe/primer melt region.

In view of the above design considerations, the present invention alsoprovides a method of designing primers and probes that are useful forthermal melt analysis of a locus of interest that contains benignvariants and disease-causing variants that are in close proximity. Inaccordance with this aspect, the method comprises selecting a locus ofinterest of a disease, in which the locus has benign variants anddisease-causing variants in close proximity. In one embodiment, closeproximity means within 3-15 nucleotides, preferably within 3-10 and morepreferably within 3-7 nucleotides of each other. The method alsocomprises designing a pair of primers for use in asymmetric PCR. Theprimers have a Tm difference of less than about 1° C. and are selectedto produce an amplicon of about 60 bp or longer. In some embodiments,the PCR produces amplicons that are between 70 and 130 bp in length. Incertain embodiments, the PCR produces amplicons that are between 80 and100 bp in length. The method further comprises designing at least twoprobes for hybridizing to one strand of the locus of interest. Thenucleotide sequence of each probe is complementary to the wild-typesequence of the locus of interest. Each probe has a Tm that is less than5° C. lower than the Tms of the primers. Each probe overlaps thenucleotide positions of the benign and disease-causing variants that arein close proximity. The probes can differ in length. In one embodiment,the probes differ in length on the 5′ end of the probe. In anotherembodiment, the probes have a 3′ end block to prevent extension. Theprobes and primers may further have the additional characteristicsdescribed herein.

According to one or more of the above embodiments, the present inventionprovides a method for detecting a disease in a patient based on thepatient's genotype. In one embodiment, diagnosticians can take advantageof a priori knowledge of benign and disease-causing variant genesequences associated with a target disease. In another embodiment, it ispossible to use the present invention as a tool to identify and genotypebenign variants and disease-causing variants associated with aparticular disease based on analysis of the target sequence and meltingsignature curves obtained by using two or more unlabeled probes. In oneembodiment, a first portion of a biological sample from a patient can besubjected to asymmetric PCR to produce a small amplicon having a locusof interest, which hybridizes to a first unlabeled probe to produce afirst melting curve. A second portion of the biological sample cansimultaneously or sequentially be subjected to asymmetric PCR to producea small amplicon having a locus of interest, which hybridizes to asecond unlabeled probe to produce a second melting curve. The unlabeledprobes have different lengths and are selected in order to produceunique melting curves in each instance.

In another embodiment, a biological sample from a patient can besubjected to asymmetric PCR to produce a small amplicon having a locusof interest. A first portion of the small amplicon having a locus ofinterest is subjected to a first unlabeled probe assay to produce afirst melting curve and a second portion of the small amplicon having alocus of interest is subjected to a second unlabeled probe assay toproduce a second melting curve. A plurality of small amplicon portionscan be subjected to unlabeled probe assays to obtain additional meltingcurves, in which unlabeled probes having different lengths are selectedin order to produce unique melting curves in each instance.

The present invention provides a method to distinguish between a benignvariant and a disease-causing neighbor variant by analyzing a firstmelting curve and a second melting curve, in which a probe meltingsignature curve of the benign variant and a probe melting signaturecurve of the disease-causing variant in the first and second meltingcurves are different. Accordingly, by reliably distinguishing between abenign variant and a disease-causing neighbor variant, the presentinvention makes it is possible to determine whether the patient has adisease-causing variant.

In another aspect, a biological sample from a patient can be dividedinto a first portion and at least a second portion. A plurality ofadditional portions may be used according to the steps described herein.In each portion, asymmetric PCR can be performed in order to produce asmall amplicon having a locus of interest in each portion. The smallamplicon having a locus of interest in each portion can be subjected toan unlabeled probe assay and high resolution thermal melt analysis inorder to produce a unique melting curve for each portion. The uniquesignature melting curves can be analyzed in order to distinguish betweena benign variant and a disease-causing neighbor variant. Accordingly, byreliably distinguishing between a benign variant and a disease-causingneighbor variant, it can be determined whether the patient has adisease-causing variant.

As described in one embodiment herein, an unlabeled probe assay includesthe steps of hybridizing an unlabeled probe to a locus of interest on asmall amplicon to form a probe-amplicon element, adding a saturated dyeto the probe-amplicon element to form a mixture, and generating amelting curve for the probe-amplicon element by measuring fluorescencefrom the dye as the mixture is heated.

The above described embodiments of a multiple unlabeled probe assay inhigh resolution thermal melt analysis are simple, fast and can be easilyadapted to microfluidic devices. The probe melting signatures of eachgenotype for targeted amplicons can be recognized using conventionalhigh resolution thermal melt software well known to the skilled artisan,including Genotype Determinator and Melt Viewer. The definitive probemelting shapes of each genotype per each mutation generated with bothprobes can be used in a clinical molecular diagnostic report. Thedefinitive probe melting shapes of each genotype per each mutationgenerated with both probes can be accepted and interpreted easily andclearly by clinicians and physicians. Probe/primer sets designedaccording to the present invention can be used to study variousdiseases, including, for example, Cystic Fibrosis. In one embodiment,the above described embodiments can be applied to a CFTR ACOG paneltesting kit. The panel recommended by The American College ofObstetricians and Gynecologists (ACOG) and the American College ofMedical Genetics (ACMG) includes 23 core mutations on CF gene.

In another aspect, the present invention provides a kit fordistinguishing between at least two variants on a target nucleic acidhaving a locus of interest and/or detecting a disease in a patient basedon the patient's genotype. Unlabeled probes designed according to thepresent invention, along with primer pairs designed according to thepresent invention can be included in a kit for performing a diagnostictest for detecting a specified disease for which the probes and primerswere designed. The kit may also be used for conducting biochemicalstudies on various nucleic acid sequences. The kit may includeinstructions for performing a diagnostic test. In a non-limitingexample, a kit can contain primer pairs, two or more unlabeled probes,as well as instructions for performing a diagnostic test for detectingvariants/mutations that are in close proximity using a target nucleicacid having a locus of interest. The kit can also contain commonreagents necessary for PCR such as polymerases, ligases, NADP, dNTPs,buffers, salts, etc. Such reagents are known to persons of ordinaryskill in the art.

Unlabeled probes and primer pairs designed according to the presentinvention can be used in any high resolution thermal melt analysisinstrument for clinical molecular diagnosis on a target disease.Unlabeled probes and primer pairs designed according to the presentinvention can also be used by clinic laboratories for clinical moleculardiagnosis of disease such as, for example, Cystic Fibrosis, Factor VLeiden genotyping, human platelet antigens, the RET proto-oncogene,lactase and hereditary hemorrhagic telangiectasia. Unlabeled probes andprimer pairs designed according to the present invention can be used asa reflexive genotyping test following HRMA scanning of a gene by cliniclaboratories for the clinical molecular diagnosis of a target disease.

The design concepts of the present invention can be applied both tosimilar close proximity variant/mutation situations on other genes ofinterest as well as to mutation discovery on other genes which havebenign variants in close proximity to disease causing variants.

EXAMPLES

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

Example 1

FIG. 4 illustrates a schematic diagram of primers, probes, and CFTR Exon10. A forward primer (400), which acts as the limiting primer, extendsin the 5′ to 3′ direction of the target nucleic acid having a locus ofinterest (410) having mutations of interest (420-424) at a locus ofinterest (430). The mutations of interest (420-424) include benignvariants I506V (420), I507V (422), and F508C (424), as well asdisease-causing variants ΔI507 (421) and ΔF508 (423). A reverse primer(440), which acts as the excess primer, extends in the 3′ to 5′direction of the target nucleic acid having a locus of interest (410).Unlabeled probe (450) having a blocker (460) at its 3′ end, has beendesigned to hybridize to the reverse strand of the target nucleic acidhaving a locus of interest (410) at the locus of interest (430).

In one non-limiting embodiment, a primer pair (F2/R4) that produces aspecific amplicon that includes the mutations of ΔI507/ΔF508 andneighboring benign variants on Exon 10 can be used according to thepresent invention. F2 has the sequence 5′-GGATTATGCCTG GCACCATTA-3′ (SEQID NO:1) and R4 has the sequence 5′-GTTGGCATGCTTTGATG ACG-3′ (SEQ IDNO:2).

In a non-limiting embodiment, two unlabeled probes (UP3 and UP4) thatproduce unique melting signature curves for the mutations of ΔI507/ΔF508and neighboring benign variants on Exon 10 can be used according to thepresent invention. The sequence of the unlabeled probes was designed tobe complementary to the wild type sequence of the region of interest onexon 10. UP3 has the sequence 5′-AAAATATCATCTTTGGTGTTTCCTATGATGAATATAG-3′ (SEQ ID NO: 3) and UP4 has the sequence5′-ATATCATCTTTGGTGTTTCCT ATGATGAATATAG-3′ (SEQ ID NO:4). In individualswith ΔF508, ΔI507 or F508C, I506V, and I507V mutations, the probe willhave a one or more base pair mismatch(es) at the corresponding locus.This design is to increase the assay sensitivity and specificity of all5 mutations/variants in the mutation(s) of interest(s) on the DNAsequence of exon 10. UP3 possesses 5 bp prior to the mutation ΔI507 andI506V, and UP4 possesses 2 bp prior to the mutation ΔI507 and I506V.

Unlabeled probes designed according to the present invention, e.g., UP3and UP4, along with primer pairs designed according to the presentinvention, e.g., F2 and R4, can be included in a kit for performing adiagnostic test for detecting a specified disease for which the probesand primers were designed. The kit may include instructions forperforming a diagnostic test. In a non-limiting example, a kit cancontain primer F2, primer R4, unlabeled probe UP3, and unlabeled probeUP4, as well as instructions for performing a diagnostic test fordetecting cystic fibrosis transmembrane conductance regulator Exon 10variants using a biological sample from a patient.

Example 2

Biological samples having a locus of interest (see details in Table 1)were incubated with limiting primer (F2), excess primer (R4), and anunlabeled probe (UP3). The two primers were provided in differentconcentrations along with buffer, dNTPs, MgCl2, LC Green Plus, andpolymerase. Parallel mixtures using the same primers and reagents, but adifferent unlabeled probe (UP4), were also prepared. Each of themixtures was loaded onto a 96-well plate on a LC 480 and subjected toasymmetric PCR. Initially, the PCRs preceded using the limiting andexcess primers to produce small amplicons. In each mixture subjected toPCR, once the limiting primer was exhausted, the unlabeled probe thenattached to the locus of interest on a small amplicon in conjunctionwith the excess primer and PCR continued. It is noted that the unlabeledprobes each had a blocker in place so as to not extend during the PCRs.

TABLE 1 NA01531 Coriell Exon 10 deltaF508 hom NA07552 Coriell Exon10/Exon 11 deltaF508/R553X Compound het NA11275 Coriell Exon 19/Exon 103659delC/deltaF508 Compound het NA11277 Coriell Exon 10 delta I507 hetNA11281 Coriell Intron 4/Exon 10 621 + 1 G > T/F508 Compound het[PHE508DEL] NA11283 Coriell Exon 9/Exon 10 A455E/deltaF508 Compound hetNA11284 Coriell Exon 11/Exon R560T/deltaF508 Compound het 10 NA13591Coriell Exon 4/Exon 10 R117H/deltaF508 Compound het NA18799 Coriell Exon13/Exon 2184delA/delta F508 Compound het 10 NA18800 Coriell Intron12/Exon 1898 + 1G > A/deltaF508 Compound het 10 NA07469 Coriell Exon11/Exon 10 1789C > T/R553X/deltaF508 Compound het NA07381 Coriell Intron19/Exon 3849 + 10kbC > T/deltaF508 Compound het 10 NA13033 Corielle Exon10 F508C Hom NA21551 Corielle Exon 10 delta F508/I507V Compound hetI506V Utah Exon 10 1648A/G Het

The PCR cycles were performed as detailed in Table 2:

TABLE 2 Unlabeled Probe Assay PCR Cycling Temperature Duration SettingCycle ° C. (seconds) Hot Start 1 95 10  PCR cycling 60 94 5 58 5 72 6Pre-melt Denature 1 94 5 & Re-nature 45 3 Melt 1 95 Ramp rate - 0.06°C./sec; 10 acquisition/° C. 45 1

After completion of the PCR cycles, the probe-amplicon products in eachportion were subjected to HRMA to produce unique melting signatures atlower temperature regions from the small amplicon Tm. The data showedthat the primer pair (F2/R4) produced the specific amplicon thatincluded the mutations of ΔI507/ΔF508 and neighboring benign variants onExon 10. The data also showed that the unlabeled Probe UP3 placedmutation ΔI507 at the 6^(th) bp position.

Experiments using the unlabeled probes, primer pairs, and target nucleicacid having a locus of interest showed that all samples were amplified.This experiment was done several times using the Roche LightCycler® 480(LC480) cycler platform and a microfluidic instrument. The results ofthe experiments are shown in FIGS. 5 and 6. FIGS. 5 and 6 illustrate aso-called “derivative plot” which describes the derivative offluorescence with respect to temperature as a function of temperature(e.g., dF/dT vs. T). FIG. 5 illustrates thermal melt curves of anamplification product generated using the primer pairs and tested withthe first unlabeled primer (UP3) on the microfluidic instrument. FIG. 6illustrates thermal melt curves of an amplification product generatedusing the primer pairs and tested with the second unlabeled primer (UP4)on the microfluidic instrument.

FIG. 5 demonstrates that UP3 clearly distinguished the homozygousvariant ΔF508, the compound heterozygote of ΔF508 and I507V,heterozygote of ΔF508, heterozygote of I506V and the wild-type. However,there is no significant difference between heterozygote of ΔI507 andF508C. Thus, the design of UP3 was found to present the meltingsignature of ΔI507 heterozygote with two melting peaks in the probemelting region, as well as definitively distinguish I506V benign variantfrom the wild-type and other mutations. However, the limitation forusing this probe alone is that the difference between ΔI507 heterozygoteand F508C heterozygote was not reliably distinguishable.

FIG. 6 demonstrates that UP4 clearly distinguished the homozygous ofΔF508, the compound heterozygote of ΔF508 and I507V, heterozygote ofΔF508, heterozygote of ΔI507, heterozygote of F508C and the wild-type.However, the I506V heterozygote was not reliably distinguishable fromthe wild-type.

Analysis of both melting signatures from the two tests were used toclearly identify and differentiate ΔF508 and ΔI507 and nearbyneighboring benign variants, F508C, I506V, and I507V on exon 10.

Example 3

Primers and probes were designed for use in a Cystic Fibrosis AmericanCollege of Obstetricians and Gynecologists (ACOG) panel. The ACOG panelincluded the primers and probes described in Examples 1 and 2, above.The sequences of these primers and probes are listed in Table 3 belowand details pertaining thereto are listed in Table 4. Forward primersare denominated with the suffix “F#,” reverse primers are denominatedwith the suffix “R#,” and probes are denominated with the suffix “UP#.”

TABLE 3  Exon Primer or Probe Name Sequence 5′-3′  3 Exon 3 G85E FlGCCCTTCGGCGATGTTTT Exon 3 G85E R1 gatccttacCCCTAAATATAAAAAG  4Exon 4 R117H F3 ATGACCCGGATAACAAGGAG Exon 4 R117H R2CATAAGCCTATGCCTAGATAAATCG Intron Exon 4 621 + 1G > T F2GAGAATAGCTATGTTTAGTTTGATTT  4 Exon 4 621 + 1G > T R3gcctgtgcaaggaagtatta Intron Exon 5 711 + 1G > T F2GTCTCCTTTCCAACAACCTGAA  5 Exon 5 711 + 1 G > T R1 agtgcctaaaagattaaatcaa 7 Exon 7 R334W F2 GCACTAATCAAAGGAATCATCCTC Exon 7 R334W R2CAGAATGAGATGGTGGTGAAT  7 Exon 7 R347P F3 CCACCATCTCATTCTGCATTGExon 7 R347P R2 GGAAATTGCCGAGTGACC  9 Exon 9 A455E Flgggccatgtgcttttcaaact Exon 9 A455E R1 gaactacCTTGCCTGCTCCA e9 A455E UP1rAACCGCCAACAACTGTCCTCTTTCTAT Intron Exon 11 1717 − 1 G > A F4AGTGACTCTCTAATTTTCTATTTTTGGTAAT 10 Exon 11 1717 − 1 G > A R5CTCTGCAAACTTGGAGATGTC e10 Exon 10 507n508 F2 GGATTATGCCTGGCACCATTAExon 10 507n508 R4 GTTGGCATGCTTTGATGACG e10 507n508 UP3AAAATATCATCTTTGGTGTTTCCTATGATGAATATAG e10 507n508 UP4ATATCATCTTTGGTGTTTCCTATGATGAATATAG 11 Exon 11 G542X F8GTTTGCAGAGAAAGACAATATAGTTCT Exon 11 G542X R7 CTCAGTGTGATTCCACCTTCT 11e11 551n553 F2 GAGAAGGTGGAATCACACTG e11 551n553 R4 cagcaaatgcttgctagacce11 551n553 UP GAGGTCAACGAGCAAGAATTTCTTTA 11 Exon 11 R560T F6AACGAGCAAGAATTTCTTTAGCA Exon 11 R560T R3 GCTTGCTAGACCAATAATTAGTTATTCACIntron  Exon 12 1898 + 1G > A F2 CCTAGATGTTTTAACAGAAAAAGAAA 12Exon 12 1898 + 1G > A R3 gcattataagtaaggtattcaaagaac 13Exon 13 2184delA Fll GTCTCCTGGACAGAAACAAAAA Exon 13 2184delA R10CCCAAACTCTCCAGTCTGTTTA 14b Exon 14 2789 + 5G > A F3 GCTGTGGCTCCTTGGAAAgtExon 14 2789 + 5G > A R1 cacaatctacacaataggacatgga Intron Exon 16 3120 +1G > A F3 CTCTTACCATATTTGACTTCATCCA 16 Exon 16 3120 + 1G > A R1catacttaacggtacttatttttacat Intron Intron 19 3849 + 10kbC > T F3aagagtcttccatctgttgcagt 19 Intron 19 3849 + 10kbC > T R3gaacatttcctttcagggtgtc 19 Exon 19 R1 162X C > T F1 ttcagATGCGATCTGTGAGCExon 19 R1 162X C > T R2 CTGTTGGCATGTCAATGAACTT 19 Exon 19 3659delC F2ATTGACATGCCAACAGAAGG Exon 19 3659delC R1 CTTGTATGGTTTGGTTGACTTG 20Exon 20 W1282X G > A F2 GTCTTGGGATTCAATAACTTTGC Exon 20 W1282X G > A R1ATCACTCCAAAGGCTTTCCT 21 Exon 21 N1303K C > G F3GAAAGTATTTATTTTTTCTGGAACATTTAGAAAA Exon 21 N1303K C > G R5CCACTGTTCATAGGGATCCAA

TABLE 4 Length Tm Amplicon Exon Primer or Probe Name (bp) Ta/Cycle (°C.) Length GC %  3 Exon 3 G85E F1 18 58/40 63 69 56 Exon 3 G85E R1 25 5532  4 Exon 4 R117H F3 20 58/40 59 58 50 Exon 4 R117H R2 25 60 40 IntronExon 4 621 + 1G > T F2 26 58/40 54 55 27  4 Exon 4 621 + 1G > T R3 20 5645 Intron Exon 5 711 + 1G > T F2 22 58/40 61 68 45  5 Exon 5 711 + 1 G >T R1 22 53 27  7 Exon 7 R334W F2 24 58/40 60 53 42 Exon 7 R334W R2 21 5743  7 Exon 7 R347P F3 21 58/40 61 52 48 Exon 7 R347P R2 18 59 56  9 Exon9 A455E F1 21 58/60 63 304 48 Exon 9 A455E R1 20 60 55 e9 A455E UP1r 2756 32 Intron Exon 11 1717-1 G > A F4 31 58/40 61 54 26 10 Exon 11 1717-1G > A R5 21 60 48 10 Exon 10 507n508 F2 21 58/60 62 90 48 Exon 10507n508 R4 20 63 50 e10 507n508 UP3 37 58 29 e10 507n508 UP4 34 57 29 11Exon 11 G542X F8 27 58/40 61 51 33 Exon 11 G542X R7 21 61 48 11 e11551n553 F2 20 58/60 55 92 50 e11 551n553 R4 20 59 50 e11 551n553 UP 2655 38 11 Exon 11 R560T F6 23 58/40 60 54 35 Exon 11 R560T R3 29 62 34Intron Exon 12 1898 + 1G > A F2 26 58/40 57 67 27 12 Exon 12 1898 + 1G >A R3 27 56 30 13 Exon 13 2184delA F11 22 58/40 58 54 41 Exon 13 2184delAR10 22 58 45 14b Exon 14 2789 + 5G > A F3 20 58/40 62 51 55 Exon 142789 + 5G > A R1 25 60 40 Intron Exon 16 3120 + 1G > A F3 25 58/40 59 5536 16 Exon 16 3120 + 1G > A R1 27 54 26 Intron Intron 19 3849 + 10kbC >T F3 23 58/40 59 62 43 19 Intron 19 3849 + 10kbC > T R3 22 60 45 19 Exon19 R1162X C > T F1 20 58/40 60 51 50 Exon 19 R1162X C > T R2 22 60 41 19Exon 19 3659delC F2 20 58/40 59 51 45 Exon 19 3659delC R1 22 57 41 20Exon 20 W1282X G > A F2 23 58/40 59 51 39 Exon 20 W1282X G > A R1 20 5845 21 Exon 21 N1303K C > G F3 34 58/40 60 58 21 Exon 21 N1303K C > G R521 61 48

As shown by the above examples, two or more unlabeled probes may be usedto provide a reliable procedure to discern between benign variants anddisease-causing variants that are close in proximity on a gene.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. In addition, recitation of ranges of values herein aremerely intended to serve as a shorthand method of referring to allseparate ranges falling within the range, unless otherwise indicated,and each separate range is incorporated into the specification as if itwere individually recited herein. For example, if the range 10-15 isdisclosed, then the ranges 10-14, 10-13, 10-12, 10-11, 11-15, 11-14,11-13, 11-12, 12-15, 12-14, 12-13, 13-15, 13-14 and 14-15 are alsodisclosed. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the invention and does not pose a limitation on the scope ofthe invention unless otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element as essentialto the practice of the invention.

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. Variations of thoseembodiments may become apparent to those of ordinary skill in the artupon reading the foregoing description. The inventors expect skilledartisans to employ such variations as appropriate, and the inventorsintend for the invention to be practiced otherwise than as specificallydescribed herein. Accordingly, this invention includes all modificationsand equivalents of the subject matter recited in the claims appendedhereto as permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the invention unless otherwise indicated herein orotherwise clearly contradicted by context.

1. A method of distinguishing between at least two nearby neighborvariants on a nucleic acid having a locus of interest comprising: (a)providing a first aliquot of said nucleic acid having a locus ofinterest; (b) incubating said first aliquot of said nucleic acid with alimiting primer, an excess primer, and a first probe that is designed tohybridize to said locus of interest on a target strand of said nucleicacid; (c) performing asymmetric PCR using said first aliquot to producean excess of amplicons corresponding to the target strand to which thefirst probe hybridizes, thereby producing a first probe element; (d)providing a second aliquot of said nucleic acid target having a locus ofinterest; (e) incubating said second aliquot of said nucleic acid targetwith said limiting primer, said excess primer, and a second probe thatis designed to hybridize to said locus of interest on the target strand,wherein said first probe differs in sequence from said second probe; (f)performing asymmetric PCR using said second aliquot to produce an excessof amplicons corresponding to the target strand to which the secondprobe hybridizes, thereby producing a second probe element; (g)generating a first melting curve for the first probe element in a firstmixture with a saturating binding dye by measuring fluorescence fromsaid dye as the first mixture is heated; (h) generating a second meltingcurve for the second probe element in a second mixture with saidsaturating binding dye by measuring fluorescence from said dye as thesecond mixture is heated; and (i) analyzing said first melting curve andsaid second melting curve to distinguish between said at least twonearby neighbor variants, wherein a melting signature curve of each ofsaid at least two nearby neighbor variants is different in said firstand second melting curves.
 2. The method of claim 1, wherein said one orboth of said first and second probes are unlabeled.
 3. The method ofclaim 1, wherein steps (a)-(f) are performed simultaneously.
 4. Themethod of claim 1, wherein steps (a)-(f) are performed sequentially. 5.The method of claim 1, wherein said first probe and said second probeeach have a sequence that is complementary to a wild-type sequence ofthe gene.
 6. The method of claim 1, wherein the limiting primer and theexcess primer are each set close to the variants to reduce the ampliconsize for high genotyping sensitivity.
 7. The method of claim 1, whereineach of said first and second probes has one or more base pairmismatches at the locus of interest.
 8. The method of claim 7, whereineach of said first and second probes has 2 to 5 base pair mismatches atthe locus of interest.
 9. The method of claim 1, wherein each of saidfirst and second probes, independently, has 2 to 5 base pairs at its5′-end prior to the locus of interest.
 10. The method of claim 1,wherein the first probe has 2 or 3 base pairs at its 5′-end prior to thelocus of interest.
 11. The method of claim 1, wherein the second probehas 5 base pairs at its 5′-end prior to the mutation.
 12. The method ofclaim 1, wherein the Tm of each of the first and second probes is lessthan about 5 degrees lower than the Tms of the limiting and excessprimers and the difference of the limiting primer's Tm and the excessprimer's Tm is less than about 1° C.
 13. The method of claim 1, whereinsaid first probe and said second probe are 34 to 37 bp in length. 14.The method of claim 1, wherein said first probe and said second probeare blocked at their 3′ ends.
 15. The method of claim 1, wherein saidlocus of interest is on a gene associated with a disorder selected fromthe group consisting of Cystic Fibrosis, Factor V Leiden, human plateletantigens, a RET proto-oncogene associated disease, lactase hemorrhagictelangiectasia, and hereditary hemorrhagic telangiectasia.
 16. Themethod of claim 1, wherein said locus of interest is Exon 11 of CysticFibrosis transmembrane conductance regulator gene.
 17. The method ofclaim 1, wherein said locus of interest is Exon 10 of Cystic Fibrosistransmembrane conductance regulator gene.
 18. The method of claim 17,wherein said limiting primer has a nucleotide sequence of5′-GGATTATGCCTGGCACCATTA-3′ (SEQ ID NO: 1).
 19. The method of claim 17,wherein said excess primer has a nucleotide sequence of 5′-GTTGGCATGCTTTGATGACG-3′ (SEQ ID NO: 2).
 20. The method of claim 17,wherein said first probe has a nucleotide sequence of5′-AAAATATCATCTTTGGTGTTTCCTATGATGAATATAG-3′ (SEQ ID NO:3).
 21. Themethod of claim 20, wherein said first unlabeled probe is blocked at its3′ end.
 22. The method of claim 17, wherein said second probe has anucleotide sequence of 5′-ATATCATCTTTGGTGTTTCCTATGATGAATATAG-3′ (SEQ IDNO: 4).
 23. The method of claim 22, wherein said second unlabeled probeis blocked at its 3′ end.
 24. The method of claim 17, wherein said atleast two nearby neighbor variants are ΔI507 and F508C.
 25. A method ofdistinguishing between at least two nearby neighbor variants on a genecomprising: (a) mixing a first portion of a target nucleic acid having alocus of interest with a first primer and a second primer, the primersconfigured for amplifying the target nucleic acid having a locus ofinterest, and a first unlabeled probe; (b) in parallel, mixing a secondportion of said target nucleic acid having a locus of interest with saidfirst primer, said second primer, and a second unlabeled probe; (c)simultaneously amplifying the target nucleic acid having a locus ofinterest to generate amplicons that hybridize to said first unlabeledprobe and to said second unlabeled probe to form a first probe elementand a second probe element, respectively, wherein said first unlabeledprobe differs in sequence from said second unlabeled probe; (d)generating a first melting curve for the first probe element in thepresence of a saturating binding dye by measuring fluorescence from saiddye as the mixture is heated; (e) generating a second melting curve forthe second probe element in the presence of said saturating binding dyeby measuring fluorescence from said dye as the mixture is heated; and(f) analyzing said first melting curve and said second melting curve todistinguish between said at least two nearby neighbor variants, whereina probe melting signature curve of each of said at least two nearbyneighbor variants is different in said first and second melting curves.26. A method of detecting a disease in a patient based on said patient'sgenotype and a priori knowledge of benign and disease-causing variantgene sequences associated with said disease, comprising: (a) obtaining abiological sample from said patient; (b) subjecting a first portion ofsaid biological sample to asymmetric PCR involving a limiting primer, anexcess primer, and a first probe to produce a first probe-ampliconelement; (c) subjecting a second portion of said biological sample toasymmetric PCR involving said limiting primer, said excess primer, and asecond probe to produce a second probe-amplicon element; (d) generatinga first melting curve and a second melting curve by subjecting saidfirst and second probe-amplicon melting elements to high resolutionthermal melting analysis, respectively; (e) distinguishing between abenign variant and a disease-causing neighbor variant by analyzing saidfirst melting curve and said second melting curve, wherein a probemelting signature curve of said benign variant and a probe meltingsignature curve of said disease-causing variant in said first and secondmelting curves are different; and (f) determining whether said patienthas a disease-causing variant.
 27. The method of claim 26, wherein saiddisease is selected from the group consisting of Cystic Fibrosis, FactorV Leiden, human platelet antigens, a RET proto-oncogene associateddisease, lactase hemorrhagic telangiectasia, and hereditary hemorrhagictelangiectasia.
 28. The method of claim 27, wherein said disease cysticfibrosis.
 29. The method of claim 26, wherein said benign and saiddisease-causing variant are neighboring variants.
 30. The method ofclaim 29, wherein said benign variant is F508C and said disease-causingvariant is ΔI507.
 31. The method of claim 26, wherein said limitingprimer has a nucleotide sequence of 5′-GGATTATGCCTGGCACCATTA-3′ (SEQ IDNO: 1).
 32. The method of claim 26, wherein said excess primer has anucleotide sequence of 5′-GTTGGCATGCTTTGATGACG-3′ (SEQ ID NO: 2). 33.The method of claim 26, wherein said first probe has a nucleotidesequence of 5′-AAAATATCATCTTTGGTGTTTCCTATGATGAATATAG-3′ (SEQ ID NO: 3).34. The method of claim 33, wherein said first probe is blocked at its3′ end.
 35. The method of claim 26, wherein said second probe has anucleotide sequence of 5′-ATATCATCTTTGGTGTTTCCTATGATGAATATAG-3′ (SEQ IDNO: 4).
 36. The method of claim 35, wherein said second probe is blockedat its 3′ end.
 37. A primer having a nucleotide sequence of5′-GGATTATGCCTGGCACCATTA-3′ (SEQ ID NO: 1).
 38. A primer having anucleotide sequence of 5′-GTTGGCATGCTTTGATGACG-3′ (SEQ ID NO: 2).
 39. Aprobe having a nucleotide sequence of 5′-AAAATATCATCTTTGGTGTTTCCTATGATGAATATAG-3′ (SEQ ID NO: 3) and being blocked at its 3′ end.
 40. Aprobe having a nucleotide sequence of 5′-ATATCATCTTTGGTGTTTCCTATGATGAATATAG-3′ (SEQ ID NO: 4) and being blocked at its 3′ end.
 41. A kitcomprising: (a) a primer having a nucleotide sequence of5′-GGATTATGCCTGGCACCATTA-3′ (SEQ ID NO: 1); (b) a primer having anucleotide sequence of 5′-GTTGGCATGCTTTGATGACG-3′ (SEQ ID NO: 2); (c) aprobe having a nucleotide sequence of 5′-AAAATATCATCTTTGGTGTTTCCTATGATGAATATAG-3′ (SEQ ID NO: 3) and being blocked at its 3′ end; (d)a probe having a nucleotide sequence of 5′-ATATCATCTTTGGTGTTTCCTATGATGAATATAG-3′ (SEQ ID NO: 4) and being blocked at its 3′ end; and (e)instructions for performing a diagnostic test for detecting cysticfibrosis transmembrane conductance regulator Exon 10 variants using abiological sample from a patient.
 42. A method of detecting a disease ina patient based on said patient's genotype and a priori knowledge ofbenign and disease-causing variant gene sequences associated with saiddisease, comprising: (a) obtaining a biological sample from saidpatient; (b) dividing said biological sample into a first portion and asecond portion; (c) performing asymmetric PCR in order to produce asmall amplicon in each of said first portion and said second portion;(c) subjecting said first portion to a first unlabeled probe assay toproduce a first melting curve; (d) subjecting said second portion to asecond unlabeled probe assay to produce a second melting curve; (e)distinguishing between a benign variant and a disease-causing neighborvariant by comparing said first melting curve and said second meltingcurve, wherein a probe melting signature curve of said benign variantand a probe melting signature curve of said disease-causing variant insaid first and second melting curves are different; and (f) determiningwhether said patient has a disease-causing variant.
 43. The method ofclaim 42, wherein said first unlabeled probe assay comprises hybridizinga first unlabeled probe to a locus of interest on said small amplicon toform a first probe element, adding a saturated dye to said first probeelement to form a mixture, and generating a first melting curve for thefirst probe element by measuring fluorescence from said dye as themixture is heated.
 44. The method of claim 42, wherein said secondunlabeled probe assay comprises hybridizing a second unlabeled probe toa locus of interest on said small amplicon to form a second probeelement, adding a saturated dye to said second probe element to form amixture, and generating a second melting curve for the second probeelement by measuring fluorescence from said dye as the mixture isheated.
 45. The method of claim 42, wherein said disease is selectedfrom the group consisting of Cystic Fibrosis, Factor V Leiden, humanplatelet antigens, a RET proto-oncogene associated disease, lactasehemorrhagic telangiectasia, and hereditary hemorrhagic telangiectasia.46. A method of designing primers and probes that are useful for thermalmelt analysis of a nucleic acid having a locus of interest that containsone or more benign variants and one or more disease-causing variantsthat are in close proximity, the method comprising: (a) selecting alocus of interest on a nucleic acid that is associated with a disease,in which the nucleic acid has one or more benign variants and one ormore disease-causing variants in close proximity on said locus ofinterest; (b) designing a pair of primers for use in asymmetric PCR,wherein the primers have a Tm difference of less than about 1° C. andare selected to produce an amplicon of about 60-120 base pairs uponamplification of the nucleic acid having a locus of interest; and (c)designing at least two probes for hybridizing to one strand of thenucleic acid having a locus of interest, wherein the nucleotide sequenceof each probe is complementary to the nucleic acid's wild-type sequence,wherein each probe has a Tm that is less 5° C. lower than the Tms of theprimers, wherein each probe overlaps the nucleotide positions of the oneor more benign and one or more disease-causing variants that are inclose proximity on said locus of interest and wherein the probes differin length.
 47. The method of claim 46, wherein the probes differ inlength on the 5′ end of the probe.
 48. The method of claim 46, whereinthe probes have a 3′ end block to prevent extension.
 49. The method ofclaim 46, wherein the length of each of said probes is about ⅓ thelength of said amplicon.
 50. A method of distinguishing between at leasttwo nearby neighbor variants on a gene comprising: (a) providing anamplicon having a locus of interest; (b) hybridizing a first unlabeledprobe to said locus of interest on a first portion of the amplicon toform a first probe element; (c) hybridizing a second unlabeled probe tosaid locus of interest on a second portion of the amplicon to form asecond probe element, wherein said first unlabeled probe differs insequence from said second unlabeled probe; (d) generating a firstmelting curve for the first probe element in a first mixture with asaturating binding dye by measuring fluorescence from said dye as thefirst mixture is heated; (e) generating a second melting curve for thesecond probe element in a second mixture said saturating binding dye bymeasuring fluorescence from said dye as the second mixture is heated;and (f) analyzing said first melting curve and said second melting curveto distinguish between said at least two nearby neighbor variants,wherein a melting signature curve of each of said at least two nearbyneighbor variants is different in said first and second melting curves.51. The method of claim 50, wherein said amplicon is produced by mixinga target nucleic acid having a locus of interest with a first primer anda second primer, the primers configured for amplifying the targetnucleic acid having a locus of interest, and amplifying the targetnucleic acid having a locus of interest to generate an amplicon.
 52. Themethod of claim 50, wherein said amplicon is produced using asymmetricPCR.
 53. The method of claim 50, wherein steps (a)-(f) are performedsimultaneously.
 54. The method of claim 50, wherein steps (a)-(f) areperformed sequentially.
 55. The method of claim 50, wherein said firstunlabeled probe and said second unlabeled probe each have a sequencethat is complementary to a wild-type sequence of the gene.
 56. Themethod of claim 50, wherein the first primer and the second primer areeach set close to the variants to reduce the amplicon size for highgenotyping sensitivity.
 57. The method of claim 50, wherein the probehas one or more base pair mismatches at the locus of interest.
 58. Themethod of claim 50, wherein the unlabeled probe has 2 to 5 base pairs atits 5′-end prior to the locus of interest.
 59. The method of claim 50,wherein the first unlabeled probe has 2 or 3 base pairs at its 5′-endprior to the locus of interest.
 60. The method of claim 50, wherein thesecond unlabeled probe has 5 base pairs at its 5′-end prior to themutation.
 61. The method of claim 50, wherein the probe Tm is less thanabout 5 degrees lower than primer Tms and the difference of the firstprimer's Tm and the second primer's Tm is less than about 1° C.
 62. Themethod of claim 50, wherein said first probe and said second probe are34 to 37 bp in length.
 63. The method of claim 50, wherein said firstprobe and said second probe are blocked at their 3′ ends.
 64. The methodof claim 50, wherein the first primer is a limiting primer in theasymmetric PCR.
 65. The method of claim 50, wherein the second primer isan excess primer in the asymmetric PCR.
 66. The method of claim 50,wherein said locus of interest is Exon 10 of Cystic Fibrosistransmembrane conductance regulator gene.
 67. The method of claim 50,wherein said locus of interest is Exon 11 of Cystic Fibrosistransmembrane conductance regulator gene.
 68. A method of detecting adisease in a patient based on said patient's genotype and a prioriknowledge of benign and disease-causing variant gene sequencesassociated with said disease, comprising: (a) obtaining a biologicalsample from said patient; (b) subjecting said sample to asymmetric PCRto produce a small amplicon; (c) subjecting a first portion of saidsmall amplicon to a first unlabeled probe assay to produce a firstmelting curve; (d) subjecting a second portion of said small amplicon toa second unlabeled probe assay to produce a second melting curve; (e)distinguishing between a benign variant and a disease-causing neighborvariant by analyzing said first melting curve and said second meltingcurve, wherein a probe melting signature curve of said benign variantand a probe melting signature curve of said disease-causing variant insaid first and second melting curves are different; and (f) determiningwhether said patient has a disease-causing variant.
 69. A method ofdistinguishing between at least two nearby neighbor variants on anucleic acid having a locus of interest comprising: (a) performingasymmetric PCR using a primer pair and a first unlabeled probe; (b)performing asymmetric PCR using said primer pair and a second unlabeledprobe, wherein said first unlabeled probe differs in sequence from saidsecond unlabeled probe; (c) generating a first melting curve forproducts produced in the asymmetric PCR using the first unlabeled probein a first mixture with a saturating binding dye by measuringfluorescence from said dye as the first mixture is heated; (d)generating a second melting curve for products produced in theasymmetric PCR using the second unlabeled probe in a second mixture withsaid saturating binding dye by measuring fluorescence from said dye asthe second mixture is heated; and (e) analyzing said first melting curveand said second melting curve to distinguish between said at least twonearby neighbor variants, wherein a melting signature curve of each ofsaid at least two nearby neighbor variants is different in said firstand second melting curves.
 70. The method of claim 69, wherein each ofsaid asymmetric PCRs in steps (a) and (b) produce PCR productscomprising small double-stranded amplicons and probe/primer amplicons.71. The method of claim 70, wherein if two neighboring mutations cannotbe clearly separated from melt signatures produced by said probe/primeramplicons, further comprising the step of using melt signatures producedby said small double-stranded amplicons to distinguish between said atleast two nearby neighbor variants.
 72. The method of claim 70, whereinmelting of the small double-stranded amplicons and the probe/primeramplicons will provide thermal melt data for each type of amplicon. 73.The method of claim 71, further comprising the step of analyzing meltdata from both the small double-stranded amplicons and from theprobe/primer amplicons to distinguish between said at least two nearbyneighbor variants.
 74. A system for distinguishing between at least twonearby neighbor variants on a nucleic acid having a locus of interestcomprising: (a) a microfluidic device comprising a plurality of sampleloading zones, each of said sample loading zones being configured tohouse a separate asymmetric PCR using a nucleic acid having a locus ofinterest; wherein said microfluidic device comprises at least two sampleloading zones that are loaded with a limiting primer, an excess primer,and an unlabeled probe; wherein at least one of said at least two sampleloading zones that are loaded with an unlabeled probe contains a firstunlabeled probe as the loaded unlabeled probe and at least one of saidat least two sample loading zones that are loaded with an unlabeledprobe contains a second unlabeled probe as the loaded unlabeled probe;(b) a HRMA device, comprising a heating element, a fluorescenceexcitation light source and a fluorescence collection aperture,configured to thermally melt probe-amplicon elements obtained fromasymmetric PCRs in said sample loading zones, and to generatefluorescence derivative melting curves for said probe-amplicon elements;and (c) a fluorescence derivative melting curve analysis deviceconfigured to compare at least two melting curves generated by said HRMAdevice so as to distinguish between at least two nearby neighborvariants on the nucleic acid having a locus of interest.